Pharmaceutical compositions and utilization thereof particularly for the treatment of neurodegenerative diseases

ABSTRACT

The present invention relates to pharmaceutical compositions that act on the p53 protein or its gene and methods of use for the treatment of neurodegenerative diseases.

The present invention relates to pharmaceutical compositions and theiruse, especially in the treatment of neurodegenerative diseases. Itrelates more particularly to the use of compounds acting on the p53protein or on its gene for the preparation of a pharmaceuticalcomposition intended for the treatment of neurodegenerative diseases.

The p53 gene encodes a nuclear protein of 53 kDa. The wild-type geneencoding the native p53 has an antioncogenic activity [for a review, seefor example Oren, FASEB J. 6 (1992) 3169]. In particular, the wild-typep53 protein is capable of inhibiting the formation of transformationfoci in the fibroblasts of rodents infected with various combinations ofoncogenes. The form mutated by deletion and/or mutation of this gene ison the contrary involved in the development of most human cancers [Bakeret al., Science 244 (1989) 217]. Its mutated forms are also capable ofcooperating with the ras oncogenes to transform murine fibroblasts. Forthis reason, the p53 protein or its gene have been widely studied astargets for the treatment of cancers. Moreover, Chopp et al. (Biochem.Biophys. Res. Com 182 (1992) 1201) have described a p53 expression inischaemic mouse brain. However, nothing indicates in these results ifthis expression constitutes a cause of neurodegeneration, or a parallelphenomenon. Furthermore, no therapeutic approach is envisaged orsuggested in this document.

The present invention partially results from the demonstration that thep53 protein constitutes a mediator of neuronal degeneration. It alsoresults from the demonstration that the use of compounds capable of atleast partially inhibiting the activity of p53 protein can make itpossible to block the process of neuronal death.

In order to study the molecular mechanisms of neuronal degeneration, theApplicant has used, as a model, mice in which the expression of the p53gene has been inactivated [Donehower et al., Nature 356 (1992) 215].Irreversible focal ischaemia experiments were performed on these mice,and the volumes of infarct were compared with those observed in controlwild-type mice (same strain, same sex, same age, same supplier). Theresults obtained showed a statistically significant decrease of 20% inthe volumes of infarct after ischaemia in mice not expressing the p53gene (cf. examples). Furthermore, the Applicant has also demonstratedthat the use of anti-p53 antisense makes it possible to reduce theglutamate-induced death on cortical cell cultures. These resultsdemonstrate that the p53 protein plays a mediating role in neuronaldegeneration, an observation which has never been reported in the priorart, and that a control of the activity of this protein makes itpossible to combat neuronal death. The p53 protein, its gene and all thefactors capable of interacting with it therefore constitute newpharmacological targets in the treatment of neurodegenerative processes.The invention therefore consists, in part, in the use of compoundscapable of at least partially blocking the activity of p53 for thetreatment of neurodegenerative diseases.

A first subject of the present invention consists in the use of acompound which at least partially inhibits the activity of the p53protein for the preparation of a pharmaceutical composition intended forthe treatment and/or the prevention of neurodegenerative diseases.

The compounds which at least partially inhibit the activity of the p53protein for the purposes of the present invention may be compounds whichact (i) on the synthesis of p53, at the transcriptional, translationalor post-translational levels, or (ii) on the binding of p53 to DNA.

Among the compounds which act on the synthesis of the p53 protein, theremay be mentioned the antisense nucleotide sequences capable of reducingor of suppressing the expression of p53, at the transcriptional ortranslational level.

Such sequences may indeed be directed against the p53 mRNA and act onits translation into protein: they may be oligonucleotides (synthetic,chemically modified and the like, as described for example inApplications EP 092 574, EP 231 495, WO 92/03568; WO 91/13080 and thelike) or DNA sequences encoding RNAs capable of selectively interactingwith the p53 mRNA, according to the technique described for example inapplication EP 140 308.

Such sequences may also be directed against the gene encoding p53 andact on its transcription into RNA. More particularly, these sequencesmay be directed against coding regions of the gene (p53 structuralgene), or against noncoding regions: regions regulating transcription,exons and the like. Such sequences can be prepared under the conditionsdescribed for example in EP 558 634, WO 91/06626, WO 92/10590, WO93/10820 and the like).

Among the compounds which act on the binding of p53 to DNA, there may bementioned more particularly p53 antagonists, or proteins capable ofinteracting with p53 and of thus modulating its DNA-binding activity. Inthis regard, there may be mentioned the negative dominant mutants of p53consisting essentially of inactive mutated form, which are capable ofentering into competition with the wild-type protein for the interactionwith DNA. Such mutants are for example the p53Val135 mutant, or otherforms described for example in Michalovitz et al. [J. Cell. Bioch. 45(1991) 22]. They can be used as such, but, preferably, they are usedwithin the framework of the present invention in the form of geneticconstructs capable of expressing these mutants in vivo. Other compoundscapable of at least partially inhibiting the binding of p53 to DNAconsist of double-stranded nucleic acids reproducing the site forbinding of p53 to DNA [El-Deiry et al., Nature 1 (1992) 45; Kern et al.,Science 252, 1708; Friedman et al., PNAS 90 (1993) 3319]. The Applicanthas indeed shown that such nucleic acids were capable of complexing thetranscription factors present in the cells, of preventing them fromattaching to their endogenous sites, and thus, of blocking theirtranscriptional activity.

In a preferred mode, the compound used within the framework of thepresent invention is a double-stranded nucleic acid comprising all orpart of the site for the binding of p53 to DNA. More preferably, thenucleic acid comprises all or part of the sequence SEQ ID No. 2 or anactive variant thereof. The term active variant designates, for thepurposes of the invention, any variant of the sequence SEQ ID No. 2which has conserved the properties of attachment to the p53 protein.Such variants can be obtained by mutation, deletion, substitution and/oraddition of bases to the sequence SEQ ID No. 2, followed by verificationin vitro of the binding activity.

In another preferred mode, the compound used within the framework of thepresent invention is a nucleic acid encoding a mutated form of the p53protein capable of antagonizing the activity thereof.

Still in a preferred mode, the compound used within the framework of thepresent invention is an antisense nucleic acid capable of reducing thelevels of expression of the p53 protein, at the transcriptional ortranslational level. More preferably, it is a DNA encoding an antisenseribonucleic acid capable of inhibiting the translation of the p53cellular mRNA. Such an antisense is represented on the sequence SEQ IDNo. 1.

The nucleic acid can be used as such, for example after injection intoman or animals, in order to induce a protection or to treat neuronaldegeneration. In particular, it can be injected in naked DNA formaccording to the technique described in application WO 90/11092. It canalso be administered in complexed form, for example with DEAE-dextran[Pagano et al., J. Virol. 1 (1967) 891], with nuclear proteins [Kanedaet al., Science 243 (1989) 375], with lipids [Felgner et al., PNAS 84(1987) 7413], in the form of liposomes [Fraley et al., J. Biol. Chem.255 (1980) 10431], and the like.

Preferably, the nucleic acid used within the framework of the inventionforms part of a vector. The use of such a vector makes it possible,indeed, to enhance the administration of the nucleic acid into the cellsto be treated, and also to increase its stability in the said cells,which makes it possible to obtain a lasting inhibitory effect.Furthermore, it is possible to introduce several nucleic acid sequencesinto the same vector, which also increases the efficiency of thetreatment.

The vector used may be of various origins, as long as it is capable oftransforming animal cells, preferably human nerve cells. In a preferredembodiment of the invention, a viral vector is used which may be chosenfrom adenoviruses, retroviruses, adeno-associated viruses (AAV), theherpes virus, and the like.

In this regard, the subject of the present invention is also anyrecombinant virus comprising, inserted into its genome, a nucleic acidencoding a mutated form of the p53 protein capable of antagonizing theactivity thereof, and/or a nucleic acid comprising all or part of thesite for binding of p53 to DNA and/or an antisense nucleic acid capableof reducing the levels of expression of the p53 protein, at thetranscriptional or translational level.

The recombinant virus according to the invention may be chosen fromadenoviruses, retroviruses, adeno-associated viruses and the like.Preferably, it is a virus capable of infecting the nerve cells, such asespecially an adenovirus. Vectors derived from adenoviruses,retroviruses or AAVs incorporating heterologous nucleic acid sequenceshave been described in the literature [Akli et al., Nature Genetics 3(1993) 224; Stratford-Perricaudet et al., Human Gene Therapy 1 (1990)241; EP 185 573, Levrero et al., Gene 101 (1991) 195; Le Gal la Salle etal., Science 259 (1993) 988; Roemer and Friedmann, Eur. J. Biochem. 208(1992) 211; Dobson et al., Neuron 5 (1990) 353; Chiocca et al., NewBiol. 2 (1990) 739; Miyanohara et al., New Biol. 4 (1992) 238;WO91/18088].

Advantageously, the recombinant virus according to the invention is adefective virus. The term “defective virus” designates a virus incapableof replicating in the target cell. Generally, the genome of thedefective viruses used within the framework of the present inventiontherefore lacks at least the sequences necessary for the replication ofthe said virus in the infected cell. These regions may be either removed(completely or in part), or made nonfunctional, or substituted by othersequences and especially by the nucleic acid. Preferably, the defectivevirus conserves nevertheless the sequences of its genome which arenecessary for the encapsulation of the viral particles.

It is particularly advantageous to use the nucleic sequences of theinvention in a form incorporated into a defective recombinantadenovirus.

There are, indeed, various adenovirus serotypes whose structure andproperties vary somewhat, but which are not pathogenic for man, andespecially non-immunodepressed subjects. Moreover, these viruses do notintegrate into the genome of the cells which they infect, and mayincorporate large fragments of exogenous DNA. Among the variousserotypes, the use of the type 2 or 5 adenoviruses (Ad 2 or Ad 5) ispreferred within the framework of the present invention. In the case ofAd 5 adenoviruses, the sequences necessary for the replication are theE1A and E1B regions.

A specific embodiment of the invention consists in a vector, especiallya viral vector, comprising at least two nucleic acids as defined above.

The defective recombinant viruses of the invention can be prepared byhomologous recombination between a defective virus and a plasmidcarrying, inter alia, the nucleic acid sequence as defined above[Levrero et al., Gene 101 (1991) 195; Graham, EMBO J. 3(12) (1984)2917]. The homologous recombination occurs after co-transfection of thesaid viruses and plasmid into an appropriate cell line. The cell lineused should preferably (i) be transformable by the said elements, and(ii) comprise the sequences capable of complementing the genome part ofthe defective virus, preferably in integrated form in order to avoidrisks of recombination. By way of example of a line which can be usedfor the preparation of defective recombinant adenoviruses, there may bementioned the human embryonic kidney line 293 [Graham et al., J. Gen.Virol. 36 (1977) 59] which contains especially, integrated into itsgenome, the left-hand part of the genome of an Ad5 adenovirus (12%). Byway of example of a line which can be used for the preparation ofdefective recombinant retroviruses, the CRIP line may be mentioned[Danos and Mulligan, PNAS 85 (1988) 6460].

Next, the viruses which have multiplied are recovered and purifiedaccording to conventional molecular biological techniques.

The subject of the present invention is also a pharmaceuticalcomposition comprising at least one recombinant virus as defined above.

The pharmaceutical compositions of the invention can be formulated fortopical, oral, parenteral, intranasal, intravenous, intramuscular,subcutaneous or intraocular administration and the like.

Preferably, the pharmaceutical compositions contain pharmaceuticallyacceptable vehicles for an injectable formulation. They may be inparticular saline solutions (monosodium or disodium phosphate, sodium,potassium, calcium or magnesium chloride and the like, mixtures of suchsalts), sterile solutions, isotonic solutions or dry compositions,especially freeze-dried compositions, which, upon addition, depending onthe case, of sterilized water or of physiological saline, allow theconstitution of injectable solutions.

The doses of nucleic acids (sequence or vector) used for theadministration can be adjusted according to various parameters, andespecially according to the mode of administration used, the relevantpathology, the nucleic acid to be expressed, or alternatively thedesired duration of the treatment. In general, as regards therecombinant viruses according to the invention, these are formulated andadministered in the form of doses of between 10⁴ and 10¹⁴ pfu/ml, andpreferably 10⁶ to 10¹⁰ pfu/ml. The term pfu (“plaque forming unit”)corresponds to the infectious power of a virus solution, and isdetermined by infecting an appropriate cell culture, and measuring,generally after 48 hours, the number of plaques of infected cells. Thetechniques for the determination of the pfu titre of a viral solutionare well documented in the literature.

Such pharmaceutical compositions can be used in man, for the treatmentand/or prevention of neurodegenerative diseases, and in particular forthe treatment and/or the prevention of neuronal degeneration associatedwith ischaemia, hypoxia, anoxia, hypoglycaemia, epileptic fits oralternatively cerebral and spinal traumas, or for the treatment and/orthe prevention of Huntington's chorea, Alzheimer's disease, Parkinson'sdisease or amyotrophic lateral sclerosis.

The present invention will be described more fully with the aid of thefollowing examples which should be considered as illustrative andnonlimiting.

LEGEND TO THE FIGURES

FIGURE 1: Inhibition of cell death induced by glutamate on primarycultures of cortical neurones by an anti-p53 antisense nucleic acid.

GENERAL CLONING TECHNIQUES

The methods conventionally used in molecular biology, such aspreparative extractions of plasmid DNA, centrifugation of plasmid DNA incaesium chloride gradient, agarose or acrylamide gel electrophoresis,purification of DNA fragments by electroelution, phenol orphenol-chloroform extraction of proteins, ethanol or isopropanolprecipitation of DNA in saline medium, transformation in Escherichiacoli and the like, are well known to persons skilled in the art and arewidely described in the literature [Maniatis T. et al., “MolecularCloning, a Laboratory Manual”, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1982; Ausubel F. M. et al. (eds), “CurrentProtocols in Molecular Biology”, John Wiley & Sons, New York, 1987].

The pBR322- and pUC-type plasmids and the phages of the M13 series areof commercial origin (Bethesda Research Laboratories).

For the ligations, the DNA fragments can be separated according to theirsize by agarose or acrylamide gel electrophoresis, extracted with phenolor with a phenol/chloroform mixture, precipitated with ethanol and thenincubated in the presence of phage T4 DNA ligase (Biolabs) according tothe recommendations of the supplier.

The filling of the protruding 5′ ends can be performed with the Klenowfragment of E. coli DNA polymerase I (Biolabs) according to thespecifications of the supplier. The destruction of the protruding 3′ends is performed in the presence of phage T4 DNA polymerase (Biolabs)used according to the recommendations of the manufacturer. Thedestruction of the protruding 5′ ends is performed by a controlledtreatment with S1 nuclease.

Site-directed mutagenesis in vitro by synthetic oligodeoxynucleotidescan be performed according to the method developed by Taylor et al.[Nucleic Acids Res. 13 (1985) 8749-8764] using the kit distributed byAmersham.

The enzymatic amplification of the DNA fragments by the so-called PCRtechnique [Polymerase-catalyzed Chain Reaction, Saiki R. K. et al.,Science 230 (1985) 1350-1354; Mullis K. B. and Faloona F. A., Meth.Enzym. 155 (1987) 335-350] can be performed using a DNA thermal cycler(Perkin Elmer Cetus) according to the specifications of themanufacturer.

The verification of the nucleotide sequences can be performed by themethod developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977)5463-5467] using the kit distributed by Amersham.

EXAMPLES Example 1 Reduction in the Volume of Infarct in Mice MadeIschaemic by Suppression of the p53 Gene

This example describes the effect of the suppression of the p53 gene onthe volume of infarct in mice made ischaemic. For that, ischaemias wereinduced in mice by occlusion of the middle cerebral artery, and thevolumes of infarct were determined and then compared.

Procedure: The animals (C57/Blc male mice 9 to 12 weeks old, Genpharm,Denmark; wild-type homozygots or Δp53) were anaesthetized in a mixtureof oxygen, nitrous oxide and 1.8% halothane, and maintained under theseconditions during the entire surgical procedure. The rectal temperatureis maintained at 37° C.±0.5 by a heating cover. The left middle cerebralartery was then cauterized by electrocoagulation by means of a bipolarclip. The wound was then stitched up and the animals placed in a room at30° C. for 24 hours, with food and drink being available ad libitum.After 24 hours, the animals were sacrificed by decapitation. The brainswere removed, immersed in an isopentane bath at −30° C. and then storedat −80° C. 40 μm histological sections were then made in a cryostat at−20° C. at the rate of one section every 500 μm, from the appearance ofthe infarction until it disappeared. These sections were then stainedwith Cresyl violet. The volume of the infarction is determined by imageanalysis. The statistical analysis is performed by means of Student's ttest for independent groups, after verification of the homogeneity ofthe various values. In the case where the various values were nothomogeneous, the Wilcoxon's nonparametric test was used.

Results: The results obtained are presented in the table below. P53 miceControl mice Total number of 45 46 animals tested Mean weight (g) 25.7325.44 Mean temperature (° C.) 36.80 37.01 Mean volume of 24.95 +/− 1.8131.54 +/− 1.86 infarct (mm³)

These results show a reduction of the order of 20% in the volumes ofinfarct after ischaemia in the mice not expressing the p53 gene. Theseresults therefore demonstrate that a suppression of p53 activity makesit possible to reduce neuronal degeneration.

Example 2 Inhibition of Cell Death Induced by Glutamate of PrimaryCultures of Cortical Neurones by an Anti-p53 Antisense Nucleic Acid

This example describes the effect of an anti-p53 antisense nucleic acidon death induced by glutamate on embryonic rat cortical neurones, inprimary culture.

Glutamate is the principal neurotransmitter exciting the central nervoussystem. However, exposure to glutamate for abnormally long periods, orto concentrations higher than the physiological concentrations can causea neuronal toxicity designated by the term excitotoxicity [Olney Adv.Exp. Med. Biol. 203 (1986) 631]. Numerous experimental arguments suggestthat this type of toxicity contributes to the neuronal degenerationassociated with ischaemia, hypoxia, hypoglycaemia, epileptic fits oralternatively to cerebral traumas [Choi, J. Neurobiol. 23 (1992). 1261].The excitotoxicity is also thought to be involved in the pathogenesis ofdiseases such as Huntington's chorea [Young et al., Science 241 (1988)981] and Alzheimer's disease [Koh et al., Brain Res. 533 (1990) 315;Mattson et al.; J. Neurosci. 12 (1992) 376]. This example shows that thetoxic effect of glutamate is partly inhibited in the presence of anantisense nucleic acid capable of reducing the levels of expression ofthe p53 protein.

Preparation and sequence of the antisense nucleic acid: The antisenseoligonucleotide was synthesized by means of an automatic nucleotidesynthesizer (Maniatis). The sequence of the oligonucleotide is asfollows: 5′-CGACTGTGAATCCTCCAT-3′ (SEQ ID No. 1).

Study of inhibition: Embryonic Wistar rat cortex cells (E17) wereisolated according to the method of Dichter [Brain Res. 149 (1978) 279],cultured in 6-well Costar plates (35 min; density 6×10⁵ cells/plate), inDMEM medium (Dulbecco's Modified Eagle Medium) containing 10 μg/mlinsulin, 10 μg/ml transferrin, 10 ng/ml sodium selenite, 10 nMprogesterone, 1 nM triiodothyronine, and stored in an oven (37° C., 5%CO2). 2 μM anti-p53 antisense nucleic acid described above were thenadded to the cultures, during the inoculation, and then on days 1 and 2.The glutamate (5 mM) was administered on day 2, at the same time as theanti-p53 antisense nucleic acid. The toxicity induced by the glutamatewas determined after 24 hours of culture, by measuring the mitochondrialactivity according to the technique described by Manthorpe et al. [Dev.Brain. Res. 25 (1986) 191].

The results obtained are presented in FIGURE 1. They show clearly thatthe anti-p53 antisense nucleic acid is capable of reducing by about 25%the toxicity induced by glutamate.

1. A method for identifying compounds which at least partially inhibitthe activity of the p53 protein, comprising the steps of: (a) treating aculture of neuronal cells sensitive to glutamate-induced excitotoxicitywith a compound so that said compound enters said neuronal cells; (b)adding an excitotoxic amount of glutamate to the culture medium of saidneuronal cells; (c) comparing the amount of excitotoxicity measured insaid neuronal cells with the amount of excitotoxicity measured inneuronal cells which were not treated with said compound
 2. The methodaccording to claim 1, wherein the neuronal cells are embryonic ratcortical neurons.
 3. The method according to claim 1, wherein thecompound is a recombinant virus selected from the group consisting ofadenovirus, adeno-associated virus and herpes virus, said recombinantvirus comprising a nucleic acid selected from the group consisting of:(a) nucleic acids encoding a mutated form of p53 which antagonizeswild-type p53-mediated neuronal cell degeneration in vitro; (b) the sitefor binding of p53 to DNA; and (c) nucleic acids encoding an antisenseRNA which inhibits expression of p53.
 4. The method according to claim1, wherein the compound is a p53 antisense oligonucleotide.
 5. Themethod according to claim 3, wherein said virus comprises two nucleicacids selected from the group consisting of: (a) nucleic acids encodinga mutated form of p53 which antagonizes wild-type p53-mediated neuronalcell degeneration, (b) the site for binding of p53 to DNA; and (c)nucleic acids encoding an antisense RNA which inhibits expression ofp53.